AGR2451 Lecture 13 No reading this week Reminder that all overheads are on Reserve and all text (incl. Lectures 1 + 2) are online Review of last lecture Nitrogen - Key Points: Slide 13.1

Lecture 13 - "Introduction to Environmental Stress: Water" 1. Plants are immobile. 2. What environmental signals does a plant need to integrate? demo 3. In agriculture, the losses due to abiotic stresses are enormous as much as 50-80% compared to maximum yields, due to drought, high soil salt, flooding, low and high temperature 4. Plants can adapt to their environment using post-embryonic development from meristems: How do plants respond to: too little light? too much light? too little water or organic Nitrogen? Slide 13.2 Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.931 ASPP, Rockville MD, 2000

How do plants respond to: too much water (flooding). Why is this a problem? Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.1180 ASPP, Rockville MD, 2000 Slide A. Water use How do land plants/agriculture lose water? In the United States, 80% of all consumptive (non-reusible) water goes to crop irrigation. Water will likely be the most limiting crop nutrient in this century and the cause of famines and wars unless solutions are found. 5B. Effect of Crops utilize water at different efficiencies = "water use efficiency". PlantKg water used per Kg Dry Matter Alfalfa850 Soybeans650 Oats, potatoes580 Wheat550 Corn350 Sorghum300 Why do corn and sorghum use water >2-fold more efficiently than alfalfa or soybeans?? Plants, Genes and Agriculture,p.190 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994

5C. How does soil type influence the amount of water available to a plant and why? Water-holding capacities of soils: Soil TypeRelative Water Availability Course sand1.25 Very fine sand3.00 Silt loam5.25 Silty clay6.50 Clay7.00 5D. Since clay holds water the best, why aren't soils that are 100% clay the best for plants? 6. Three very important plant stresses are drought, freezing and high soil salt. Salt is left behind by solutes dissolved in irrigation water and left behind by evaporation and as part of applied fertilizers. How does each of these stresses cause problems for plants? a) drought? b) freezing? demo with sugar c) soil salinity? demo on board Remarkably, therefore, each of these apparently separate plant stresses create the same end-effect for a plant cell cytoplasm --- this is cytoplasmic dehydration. Plants, Genes and Agriculture,p.198 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 Slide 13.4

Effects of Water Stress A high water content is crucial to a cell. When a plant cell becomes dehydrated (water content down to ~23%) or even worse, dessicated (below 23%), proteins and intracellular lipid membranes and the plasma membrane become severely damaged. Why? a) Proteins? Therefore, water is crucial to permitting macromolecular structures such as membranes to form, proteins to fold correctly or protein-protein interactions to occur, because all of these rely on the hydrophobic effect and/or correct surface interactions with the polar charge on water molecules. b) Membranes? demo with oil and water Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.4 ASPP, Rockville MD, 2000 Slide 13.5 Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.437 ASPP, Rockville MD, 2000

. Adaptations to Drought, Freezing and Salinity Problem -too high of a solute concentration being formed outside of the cell (in the apoplast or soil). draw on board Causes water to flow from a compartment of low solutes (inside the cell) to a compartment of a high solutes (outside the cell) until the water potential is balanced. Consequence - concentration of charged solutes inside the cell (Na+, K+, etc.) increases and these can interact with the surface of proteins and damage their folding. The cytoplasm sap becomes viscous due to an increased concentration of proteins and organic molecules, causing deleterious interactions. Plants respond ("tolerate") to these problems in two ways: a) Produce organic molecules such as sugars, the amino acid proline, and sugars with hydroxyl OH groups (alcohols). Slide 13.6 The key feature shared by these molecules is that they are neutrally charged, but they are polar molecules that form hydrogen bonds. Why is this important? Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.1165 ASPP, Rockville MD, 2000

Sugars or proline can satisfy the hydrogen bonding requirement of polar groups on the surface of a protein and thus take the place of water (they form a a "replacement water" layer on the surface of proteins) These molecules are osmolytes. They increase the solute concentration of the cytoplasm but do not interfere in normal metabolism (unlike Na+ or K+ solutes). "Osmotic adjustment" is therefore a strategy used by plant cells to protect proteins and membranes. How do they do this?? Protein folding is protected Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.1166 ASPP, Rockville MD, 2000 Slide 13.7

Sugars and prolines protect lipid bilayer membranes from damage during cytoplasmic dehydration F.Hoekstra, E.Golovina, and J. Buitink (2001) Mechanisms of plant dessication tolerance. Trends in Plant Sci. 6, Elsevier Science Publishers, London,UK Slide 13.8

Second adaptation to cytoplasmic dehydration b) Since the cytoplasm still becomes dessicated, the concentration of unwanted charged ions (K+, Na+) might still be disruptive. Therefore, plant cells actively pump these ions into the vacuole or out of the cell. Vacuoles can constitute 90% of the volume of a plant cell and can be used as the "garbage can" of the cell to store toxic compounds or solutes. How? Slide 13.9 Some plants that have adapted to high salinity use active membrane transporters in root cells to transport salts into their cytoplasms. Why? Images from Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.1164 and 1166 ASPP, Rockville MD, 2000

8. The molecular mechanisms of drought, salt and freezing tolerance. Sketch = old handout i) By an unknown mechanism, low temperatures causes an influx of Ca++ from extracellular stores. ii) Ca++ inhibits phosphatase, which leads to phosphorylation of one or more protein secondary messengers. iii) Within 15 minutes of cold induction, these processes switch on the transcription of genes encoding transcription factors called CBFs. CBF transcription factors bind to a short promoter sequence (CCGAC) of suites of genes. iv) Genes which have promoters with the CCGAC sequence are transcriptionally activated. What do these genes and their protein products do? a) These proteins protect intracellular and plasma membrane proteins from freezing damage (stabilize lipid structures). b) Encode enzymes that shunt carbon/nitrogen reserves to produce proline and sugars that prevent cell dehydration during freezing. Therefore, in advance, if the cells produce extra intracellular solutes, then water will not flow out and thus prevent dehydration. ***Therefore, if a plant receives a prior cold stimulus (few days), because they produce these protectants they become "cold-acclimated" to a future freezing stress. Cold-acclimation is extremely important to crops grown in Canada. Slide Biochemistry and Molecular Biology of Plants W.Gruissem, B. Buchanan and R.Jones p.1177 ASPP, Rockville MD, 2000

v) Evidence: In the eskimo1 plant mutant of Arabidopsis, plants have increased freezing tolerance without cold acclimation: these plants have a 30-fold higher level of proline, and a 2-fold higher level of total soluble sugars than nonacclimated wild-type plants. Gilmour et al. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology 124, ASPP Publishing, Rockville, MD. Jaglo-Ottosen et al. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, AAAS Press, Washington, DC Slide vi) Evidence: Arabidopsis and Canola plants that overexpress the CBF transcription factors are more freezing, drought and salt tolerant.

vii) In dehydration stress, different parts of the plant must communicate. For example, the stomates must close. The hormone ABA (abscissic acid) is known to be involved in dehydration cell-to-cell signalling (cold, drought, salt-stress), including closing stomates. 9. This mechanism of osmotic adjustment and protectants are used under severe conditions as a normal part of the life cycle of plants and during reproduction: Explain how each of the following is related to cytoplasmic dehydration: Perennial fruit or Maple trees during winter? Sorghum under drought conditions? Pollen? Seeds? Pollen grains and seeds are both dessicated (down to 13% water content in seeds). Pollen germinates a pollen tube upon the addition of water by stigmatic cells (upon correct pollen-stigma recognition). Thus, common mechanisms between stress tolerance and evolution of pollen and seeds to be dormant: i) during gamete production to allow for free airborne and insect-borne pollen to develop (from flowers) ii) after grain-fill to permit the embryo to survive harsh conditions in a dormant (dehydrated) state, that is, to form a seed. 10. Indirect ways to save water: -preventing pathogens and pests (>50%) loss of plants -preventing post-harvest losses (fruit ripening, poor storage) (< 30%) 11.Ultimate problem with water is that land plants cannot use salt water. i) make crop plants grow with saltwater or even in the seas ii) create sea/ocean algae which produce seeds and fruits (these were adaptations to land reproduction) iii) develop a cheap technology to desalinate saltwater Slide 13.12